Molecular diagnostics comprise the detection of molecular compounds useful to identify diseases, species, individuals, etc. These molecular compounds can be, for example, ions, sugars, metabolites, fatty acids, amino acids, nucleic acids, proteins, or lipids. Nucleic acid testing (NAT) comprises the identification of specific nucleic acids from pathogens, or the identification of specific nucleic acid sequences related to diseases such as cancer, genetic diseases, genetic signature of species or individuals or markers for personalized medicine. NAT protocols often start with a sample preparation step where cells are lysed to free their nucleic acids. The nucleic acids are then specifically prepared in order to be ready for a target amplification procedure such as for example polymerase chain reaction (PCR) or isothermal amplification Recombinase Polymerase Amplification (RPA) or other nucleic acid amplification methods. Target amplification produces amplicons which can be analyzed in real time, meaning during the amplification, or at the end of the amplification in an agarose gel or on a microarray for example. Amplification procedures also exist for amplifying a signal generated by the detection of the analyte and these signal amplification approaches can also be associated with target amplification procedures. These technologies require complex protocols carried out by highly qualified personnel in dedicated facilities. For these reasons, not all laboratories, hospitals or healthcare facilities can run molecular diagnostics.
There is a need to automate complex molecular diagnostic protocols. Some approaches rely on high-throughput robotic units which are usually very expensive and can require a lot of space. There is a growing need to develop more compact instruments and mobile instrumentations such as Point-of-Care (POC) diagnostics and to miniaturize and integrate the steps of an assay—from sample preparation to answer—onto a single disposable device (ex: lab-on-a-chip devices or micro Total Analysis Systems: μTAS).
One of the main difficult steps to integrate into a disposable microfluidic system is sample preparation. Sample preparation usually starts with a cell lysis step which can be chemical and/or mechanical. Then to remove or at least control potential inhibitors of the testing process, nucleic acids can be purified. The most common techniques used to purify nucleic acids are based on solid-phase adsorption of the nucleic acids under specific conditions of pH and salt. Enzymatic reaction inhibitors such as proteins, metals and other molecules are washed away from the nucleic acids adsorbed onto the solid phase. Nucleic acids are then recovered from the solid phase by using an appropriate elution solution. The whole process requires different solutions, which need to be stored and released, a solid phase matrix and different reaction chambers. This complicates the process to integrate into a compact disposable microfluidic cartridge.
In the development of fluidic devices, there is a need to displace fluids in and out of the different processing areas in a controlled manner. Pumping and valving components are usually used.
Some have developed fluidic units enabling the automation of molecular diagnostics. For example, there exists a sample preparation cartridge with a rotary valve and a piston pump to move the fluids in the different reservoirs. There also exists mechanical lysis using ultrasounds and hard particles. Other devices use a flexible plastic assembly to lyse cells and transfer the fluids between container sectors by compressing the flexible material at a specific location. These fluidic units require several actuators to be able to perform the tasks.
The use of centripetal platforms provides a simple and effective format for the implementation of pumping and valving options. When spinning, centrifugally-induced fluid pressure causes fluid flow inside the fluidic device.
Centripetal pumping provides many advantages over other alternative pumping methods such as a syringe, piston, peristaltic, or electro-osmotic pumping. Centripetal pumping has lower electrical power requirements (the only active actuation being that needed for rotation), is independent of fluid pH or ionic strength, and does not need any external fluidic interconnections or tubing. Consequently, different assay steps requiring different sample and buffer properties (e.g., surface energy, pH) can be combined into a single fluidic centripetal device.
Another advantage of centripetal pumping is that a valve can be implemented by the geometric design of the fluidic microchannels in such a way that capillary forces balance the centripetal force due to disc rotation. By designing microfluidic structures with capillary valves of different shapes and at different positions relative to the fluidic centripetal device rotation center, the liquid flow can be interrupted and resumed by controlling the rotational speed.
Since most analytical processes for biological material require several steps, passive valving may be difficult to implement robustly. For more robustness, there is a need to implement active valves in a centripetal device. For example, it is possible to block a microfluidic channel using a phase-change material such as paraffin wax plug. This valve type is independent of the rotational speed and can be actuated by heat. For example, a plug of heat generating particles and phase-change materials can also be used. The particles absorb the electromagnetic waves from an external device (e.g. laser, IR lamp) and the phase-change material melts with the heat generated by the particles. Phase-change material valves have been described to block a fluidic channel (U.S. Pat. No. 7,837,948) and used on a centripetal nucleic acid testing device (Publ. EP 2375256).
Some active valve approaches for centripetal devices are based on actuation by an electromagnetic wave. For example, a valve closure at a desired location can be opened without contact through laser ablation and without piercing the external layer of the microfluidic device (see for example Publ. EP 1930635, PCT Pat. Appl. Publ. No. WO2004/050242, US Pat. Appl. Publ. US 2009/0189089, U.S. Pat. No. 7,709,249, U.S. Pat. No. 7,323,660).
The actuation of a phase-change material valve can be done by using electrodes which form a resistive heater onto the substrate itself. The electrodes generate heat at a specific region of interest in the microfluidic network to melt the phase-change material.
There still remains a need for an improved fluidic centripetal device with sample flow control.